This application is a National Phase application under 35 U.S.C. xc2xa7371 of International Application Number PCT/EP00/10085, filed Oct. 11, 2000 which claims priority to European Application Number 99870212.0 filed Oct. 15, 1999.
The present invention relates to new therapeutic agents with a protective effect on proteinic complexes of the inner mitochondrial membrane and that can be used preferably for preventing and/or treating partial or total ischemia, pathologies associated with ischemia or associated with mitochondrial deficiencies, or apoptosis.
Ischemia is an interruption of the blood irrigation of an organ or a tissue through artery obliteration, compression or spasma. The consequences depend on the nature of the tissue being deprived of oxygenated blood and the phenomenon duration. Total ischemia leads to tissue necrosis in a period of time variable according to the oxygen needs. Partial ischemia causes either a reduction of the organ capabilities (psychic slowing down, hepatic or kidney function insufficiency, etc.) or the incapability to modify its work at any stimulation, showing then signs of pain (angina pectoris crises, arteritis cramps). Ischemia is very deleterious for the nerve tissue. It is first to be seen in reversible metabolic and functional troubles that can lead to an abnormal nervous activity, for example, an epilepsy crisis or some cerebral cortex ischemic lesions. After a few minutes of ischemia, irreversible lesions of the nervous tissue appear, accompanied with cellular body swelling followed by necrosis thereof. This is called cerebral infarction.
It is known to use agonist molecules for alpha-adrenergic receptors and agonist molecules for alpha-pre-synaptic receptors for treating ischemia. Such compounds are used at the heart level for treating angor, which is a clinical expression of acute myocardial ischemia and the result of a temporary imbalance between the myocardium oxygen demand and the oxygen intake through circulation, leading possibly, in serious cases, to a myocardial infarction.
The use of various plant extracts for treating and/or preventing ischemia or pathologies associated with ischemia or an energy deficiency is described in International Patent Application WO 98/51291. This document also describes the working mechanism of said molecules that allow an inhibition of the activation cascade of the endothelial cells induced by hypoxia as well as the protective effect of such products on some mitochondrial deficiencies, particularly with a protective effect of the mitochondria complexes I and III. This document gives a biochemical model making it possible to obtain in vitro a protective effect from anti-ischemic compounds.
The Publication by Ukrainets I. et al. (Tetrahedron Letters Vol. 50, nxc2x0 34, p. 10331-10338 (1994)) describes derivatives and the malonic acid synthesis, particularly an ethyl malonic acid ester and symmetrical diamylides.
Malonic acid derivatives and their use for delaying plant growth are described in International Patent Application WO 87/05898.
The Publication by Venuti et al. (J. of Med. Chem. Vol. 31, p. 2145-2152 (1988)) describes, by way of an intermediate compound, a malonic acid derivate involved in the synthesis of compounds being used as prodrugs.
The Publication by Oumar-Mahamat et al. (Tetrahedron Letters Vol. 30, nxc2x0 3, p. 331-332 (1989)) describes cyclic malonic acid derivates.
Patent Application EP-A-0,099,091 describes hexahydrodioxypyrimidine derivates, the production method thereof and their use as antiviral, anti-bacterial and antitumor compounds.
Patent Application FR-2,539,412 describes 5-fluorouracyl derivates and their therapeutic use as carcinostatic agents.
The Publication by Holwood et al. (J. of Med. Chem. Vol. 10, nxc2x0 5, p. 863-867 (1967)) describes as local anesthetics, various derivates, more particularly 2-ethoxycarbonyl-aceto-2xe2x80x2,6xe2x80x2-xylidide or its corresponding amide.
The Publication by McLaughlin et al. (J. of Neurochem. Vol. 70, p. 2406-2415 (1998)) describes the effect of dopamine on the mitochbndrial inhibition compared to that of methylmalonate.
Patent Application WO97/16184 describes methods for treating ischemia comprising the step of administrating an acyl-co-enzyme A cholesterol O acyltransferase (ACAT) inhibitor such as dodecyl N-(2,6-diisopropylphenyl)-2-phenyl-malomanic acid ester and a HMG-co-enzyme A reductase inhibitor.
The aim of the present invention is to provide new compounds designed to induce a protective effect on protein complexes of the inner mitochondrial membrane and designed to be used particularly for preventing or treating partial or total ischemia, pathologies associated with ischemia or pathologies associated with mitochondrial deficiencies.
A particular aim of the present invention is to provide compounds with an improved activity and/or with no side effects of molecules in the state of the art.
The present invention relates particularly to new compounds of formula I, the salts thereof and prodrugs of such compounds: 
wherein:
R1 is CH2, NH or a ligand, preferably a carbon atom, forming together with R3 and/or R4 an aromatic or non-aromatic ring having 5 or 6 carbon atoms (n=1 or 2), optionally comprising on each ring an heteroatom, preferably a nitrogen, oxygen or sulfur atom;
R3 and/or R4 represent:
an amine with the formula: 
a group having the formula xe2x80x94Zxe2x80x94R6 wherein:
Z represents O or S,
R5 represents H or an alkyl group with 1 to 6 carbon atoms (preferably, R5 represents a methyl or ethyl group),
R6 represents an hydrophobic group, preferably selected amongst the group consisting of a tert-butyl group, an allyl group or a (aromatic or non aromatic) ring having 5, 6 or 7 carbon atoms optionally comprising one or more heteroatoms (preferably N, O or S) and wherein the carbon atoms are optionally substituted for by:
an alkyl group R7 having 1 to 6 carbon atoms (preferably, a methyl or ethyl group),
a ketone group,
a hydroxyl group,
an ester group,
a halogen element (F, Cl, Br or I),
a trifluoromethyl (CF3) group, or
a group having the formula: 
wherein:
W represents CH or a nitrogen atom,
Y represents S or O, and
R8 represents an alkyl group having 1 to 6 carbon atoms (preferably a methyl or ethyl group),
with the additional condition that, if R3 forms together with R1 said above-mentioned ring or if R3 is either an amine of formula: 
R4 represents an alkyl group (preferably a propene) having 1 to 10 (preferably 1 to 6) carbon atoms, either saturated or unsaturated, optionally comprising one or more heteroatoms (preferably N, S or O). Additionally, in said alkyl group, one or more carbons can optionally be substituted for by an halogen element (fluorine, chlorine, bromine or iodine) or a trifluoromethyl group (CF3).
In the compounds having formula I, R3 and R4 preferably represent an amine having the formula: 
a group having the formula xe2x80x94Zxe2x80x94R6 wherein R5 represents H or a methyl group, Z represents O or S (preferably O) and R6 represents a cyclopentene, a cyclohexane or a benzene group.
According to the invention, are excluded as compounds already described in the state of the art, compounds having the formula I wherein R1 is CH2, R3 is a compound having the formula Zxe2x80x94R6 wherein Z represents O, R6 represents an ethyl group, R3 is an amine having the formula NHxe2x80x94R6xe2x80x2 wherein R6xe2x80x2 represents a cyclic compound (optionally 2-substituted with an hydroxyl group) and compounds having formula I wherein R1 is CH2, R3 and R4 are amines having the formula NHxe2x80x94R6 wherein R6 represents a ring having 6 carbon atoms 2-substituted by an hydroxyl group.
Also excluded from the invention, as already described compounds, are the compounds described in tables 1, 2, and A in the Application WO87/05898, the dodecyl N-(2,6-diisopropylphenyl)-2-phenyl-malonamic acid ester and the compound having the formula I wherein R1 is CH2, R4 is a group having the formula Zxe2x80x94R6 (Z represents oxygen and R6 represents an alkyl group) and wherein R3 is an amine of formula II: 
wherein X represents O, S, CH2 or Cxe2x95x90O and derivatives having formula: 
wherein R1 represents a C1-C6 alkyl group, A represents a saturated or unsaturated ring having 3 to 7 carbon atoms; each of R2, R3 and R4 represents, independently, a hydrogen or halogen atom or a group such as C1-C6 alkoxy, C1-C6 alkyl, C1-C6 alkylthio, C1-C6 alkoxy-carbonyl, carboxyl, carboxamido, sulfonic acid, sulfonamido, acylamino, sulfonylamino, C1-C6 alkylsulfonyle, nitrile, nitro, acyloxy, phenyl or methylene-dioxy groups and each of m and n represents an integer from 0 to 4. 
wherein:
R1 represents an alkyl group, R2 is hydrogen or an alkyl group. 
wherein Zxe2x95x90CO methyl, COO methyl, or COO alkyl 
Zxe2x95x90CO2CH3, CO2C2H3 or CO2H 
wherein Xxe2x95x90CON H and Yxe2x95x90H.
According to the invention, the aromatic or non aromatic rings optionally comprise one or more heteroatoms, preferably one or more oxygen or sulfur atoms, and wherein the carbon atoms are substituted for either by ketone, hydroxyl or ester groups or by an halogen element such as described above.
The preferred compounds according to the invention are selected from the group comprised of dicyclopenten-2-yl malonate, 2-propenyl-3-oxa-2-oxobicylo[3.1.0]hexane-1-carboxylate, ethylcyclopenten-2-yl malonate, methylcyclopenten-2-yl malonate, N,Nxe2x80x2-diphenyl malonamide, ethyl-N-phenylamido ethanoate, ethyl-2,6-dimethyl-N-phenylamido ethanoate, ethyl-N-methyl-N-cyclohexylamido ethanoate and dicyclopentene-2-yl malonate, optionally their salts or prodrugs thereof.
The present invention also relates to pharmaceutically acceptable non-toxic acid addition salts of compounds according to the invention having an amine. Examples of pharmaceutically acceptable acids may include mineral acids, such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric acids, etc. and organic acids such as. acetic, citric, tartaric, benzoic, salicylic, maleic acids, etc.
The present invention also relates to the first application of compounds having formula I as a drug, as well as their salts and prodrugs of said compounds, except for the following known compounds: the N-(2,6-diisopropyl-phenyl)-2-phenyl-malonamic acid dodecyl ester and the compounds having formula I wherein R1 is CH2, R4 is a group having formula Zxe2x80x94R6 wherein Z represents oxygen and R6 represents an alkyl group and wherein R3 is an amine of formula II: 
wherein X represents O, S, CH2 or Cxe2x95x90O and the derivatives having formula: 
wherein R1 represents a C1-C6 alkyl group, A represents a saturated or an unsaturated ring having 3 to 7 carbon atoms; each of R2, R3 and R4 represents, independently, a hydrogen or halogen atom, or a group such as C1-C6 alkoxy, C1-C6 alkyl, C1-C6 alkylthio, C1-C6 alkoxycarbonyl, carboxyl, carboxamido, sulfonic acid, sulfonamido, acylamino, sulfonylamino, C1-C6 alkylsulfonyl, nitrile, nitro, acyloxy, phenyl or methylene-dioxy groups and each of m and n represents an integer from 0 to 4. 
wherein R1 represents an alkyl group, R2 hydrogen or an alkyl group.
According to the invention, it is meant by xe2x80x9cprodrugsxe2x80x9d functional derivatives of formula I compounds that may be converted, preferably in vivo, in the patient depending on the required formula I form. Such prodrugs of these compounds can be obtained using methods well-known to the man of the art such as those described in the document entitled: xe2x80x9cDesign and Prodrugsxe2x80x9d, Ed. H. Bundgaard, Elsevier (1985). The compounds according to the invention relate to two enantiomeric forms, the various separate isomers or a mixture thereof.
The new compounds according to the invention are advantageously used in order to obtain a protective effect on the protein complexes of the inner mitochondrial membrane of a mammalian""s cells, preferably a human being. The compounds according to the present invention are more particularly applied to prevent and/or treat partial or total ischemia, pathologies associated with ischemia or pathologies associated with mitochondrial deficiencies.
It is meant by xe2x80x9c(partial or total) ischemia, pathologies associated with ischemia and pathologies associated with mitochondrial deficienciesxe2x80x9d, diseases, preferably vascular ones, selected amongst the group consisting of myocardial infarction, cerebral ischemia, chronic veinous insufficiency, arteriopathies, i.e. lesions due to atherosclerosis affecting patients"" arteries, more particularly those of the lower limbs, the Raynaud""s phenomenon linked to vasospasms, leading to an artery vasoconstriction, ulcers, gangrene, alteration of the capillary permeability, capillary fragility, cicatrizations, skin alterations, retinal defects from ischemic origin, decrease of the auditive acuity from ischemic origin, troubles associated with stays in high altitude regions, angina pectoris caused by short periods of coronary obstruction, pulmonary hypertension, hepatic ischemia, Parkinson disease, myopathies and syndroms associated with vascular problems, such as diabetes, where a hypertension and an alteration of the blood flow appear in the lower limbs. Such pathologies and diseases linked to ischemia are well-known to the clinicians and practitioners who are able to adapt the use of the pharmaceutical composition for treating and/or preventing symptoms and dysfunctions of the human or animal body associated with the above-mentioned diseases and/or for preventing or reducing the possibility of being affected by them.
The Applicants have unexpectedly found out that such various syndroms or diseases can be treated using the compounds of the invention, their optional salts and prodrugs thereof, and that such compounds act according to the same biochemical action mode.
Another aspect of the present invention relates to a pharmaceutical composition comprising an appropriate pharmaceutical carrier or excipient and a sufficient amount of one or more compounds according to the invention, i.e. an amount sufficient to at least improve or prevent the above-mentioned symptoms in a mammalian, more particularly a human being. Such a sufficient amount may vary depending on some factors such as the condition of the animal to be treated, the administration route, the side-effect severity, the compound stability in the circulating serum or blood, etc. Preferably, the compound sufficient amount to be used in the pharmaceutical composition of the invention is in the range between 0.1 and 200 mg/patient, more preferably between 1 and 50 mg/patient, most preferably between 20 and 30 mg/patient, this amount being optionally able to be adapted particularly depending on the necessary administration doses and the patient""s weight, as this may be extrapolated from in vivo application examples, as disclosed hereinafter.
The pharmaceutical composition comprises an appropriate pharmaceutical carrier that can vary according to the administration mode and can be possibly combined with an adjuvant, so as to improve the therapeutic properties of the compound according to the invention or to reduce its possible side effects. Such appropriate pharmaceutical carriers or adjuvants are well-known to the man of the art and can be prepared following the procedures generally applied by chemists and may comprise any non toxic pharmaceutical carrier, either solid (including in powder form), liquid (solutions, suspensions, emulsions, etc.) or gaseous. The active pharmaceutical compound percentage (generally in the range between 5% and 70% in weight) may vary depending on the administration frequency and the possible side-effects on animal, including on human being.
In order to prepare such pharmaceutical compositions in the form of tablets, granules, capsules or tablets, suspensions, etc., it is possible to incorporate elements such as corn starch, lactose, sucrose, sorbitol, talcum, stearic acid, magnesium stearate, gums or diluents comprising a variable percentage of water or a solvent such as a syrup, oil or water suspensions, perfumed emulsions, etc. Dispersible agents used in aqueous compositions may comprise gums, alginates, dextrans, carboxymethyl cellulose derivatives, etc.
The present invention also relates to a therapeutic or preventive treatment method for ischema or pathologies associated with ischemia as above-mentioned, which includes administering a sufficient amount of formula I compound or of the pharmaceutical composition of the invention to said patient, preferably a human being, so as to prevent, reduce or eliminate the symptoms of ischemia, of pathologies associated with ischemia or associated with mitochondrial deficiencies, or apoptosis, with the additional condition that the compound is neither the dodecyl N-(2,6-diisopropyl-phenyl-)-2-phenyl-malonamic acid ester nor a formula I compound wherein R1 is CH2, R4 is a group having the formula xe2x80x94Zxe2x80x94R6 wherein Z represents oxygen and R6 represents an alkyl group and wherein R3 is an amine having formula II: 
wherein X represents O, S, CH2, Cxe2x95x90O.
A last aspect of the present invention relates to using such compounds or the pharmaceutical composition of the invention for preparing a drug for treating or preventing ischemia, pathologies associated with ischemia or associated with mitochondrial deficiencies, such as above-mentioned, or apoptosis, with the additional condition as mentioned in the preceding paragraph.
The present invention also relates to a method for preparing compounds of the invention having formula I, comprising the following steps:
esterification of an intermediate compound R3xe2x80x94H by a group of the following formula: 
in the presence of dicyclohexyl carbodiimide (DCC),
optionally followed by one or more cyclizing reactions in the presence of an oxidizing agent such as manganese acetate Mn(III) or monohydrate copper acetate Cu(II), and optionally sodium acetate.
The definitions of the above-mentioned moieties are those given in formula I for the compounds of the invention.
The various aspects of the present invention are described more in details in the non limitative examples presented hereinafter.